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Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control

BACKGROUND: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact...

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Autores principales: Katayama, Shintaro, Skoog, Tiina, Jouhilahti, Eeva-Mari, Siitonen, H. Annika, Nuutila, Kristo, Tervaniemi, Mari H, Vuola, Jyrki, Johnsson, Anna, Lönnerberg, Peter, Linnarsson, Sten, Elomaa, Outi, Kankuri, Esko, Kere, Juha
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480911/
https://www.ncbi.nlm.nih.gov/pubmed/26108968
http://dx.doi.org/10.1186/s12864-015-1671-5
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author Katayama, Shintaro
Skoog, Tiina
Jouhilahti, Eeva-Mari
Siitonen, H. Annika
Nuutila, Kristo
Tervaniemi, Mari H
Vuola, Jyrki
Johnsson, Anna
Lönnerberg, Peter
Linnarsson, Sten
Elomaa, Outi
Kankuri, Esko
Kere, Juha
author_facet Katayama, Shintaro
Skoog, Tiina
Jouhilahti, Eeva-Mari
Siitonen, H. Annika
Nuutila, Kristo
Tervaniemi, Mari H
Vuola, Jyrki
Johnsson, Anna
Lönnerberg, Peter
Linnarsson, Sten
Elomaa, Outi
Kankuri, Esko
Kere, Juha
author_sort Katayama, Shintaro
collection PubMed
description BACKGROUND: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs’ lifecycle has been omitted. RESULTS: We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types. CONCLUSIONS: The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1671-5) contains supplementary material, which is available to authorized users.
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spelling pubmed-44809112015-06-26 Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control Katayama, Shintaro Skoog, Tiina Jouhilahti, Eeva-Mari Siitonen, H. Annika Nuutila, Kristo Tervaniemi, Mari H Vuola, Jyrki Johnsson, Anna Lönnerberg, Peter Linnarsson, Sten Elomaa, Outi Kankuri, Esko Kere, Juha BMC Genomics Research Article BACKGROUND: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs’ lifecycle has been omitted. RESULTS: We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types. CONCLUSIONS: The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1671-5) contains supplementary material, which is available to authorized users. BioMed Central 2015-06-25 /pmc/articles/PMC4480911/ /pubmed/26108968 http://dx.doi.org/10.1186/s12864-015-1671-5 Text en © Katayama et al. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Katayama, Shintaro
Skoog, Tiina
Jouhilahti, Eeva-Mari
Siitonen, H. Annika
Nuutila, Kristo
Tervaniemi, Mari H
Vuola, Jyrki
Johnsson, Anna
Lönnerberg, Peter
Linnarsson, Sten
Elomaa, Outi
Kankuri, Esko
Kere, Juha
Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control
title Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control
title_full Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control
title_fullStr Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control
title_full_unstemmed Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control
title_short Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control
title_sort gene expression analysis of skin grafts and cultured keratinocytes using synthetic rna normalization reveals insights into differentiation and growth control
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480911/
https://www.ncbi.nlm.nih.gov/pubmed/26108968
http://dx.doi.org/10.1186/s12864-015-1671-5
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